DESCRIPTION (Applicant's abstract): Protein glycosylation is an essential post-translational modification in eukaryotes. The specific aims of this proposal are to determine the structure, function and catalytic mechanisms of several enzymes involved in yeast and fungal glycoprotein biosynthesis. The first enzymes are Class I al,2-mannosidases that have been conserved in all eukaryotes. These enzymes participate in endoplasmic reticulum quality control and glycoprotein folding, and are essential for complex N-glycan formation in mammalian cells. The three-dimensional structure of the endoplasmic reticulum processing al,2-mannosidase from S. cerevisiae that trims Man9GLcNAc2 to Man8GlcNAc2 was recently determined. The residues involved in catalysis and those that determine the specificity of this enzyme will be studied by mutagenesis and by X-ray crystallography of enzyme/substrate and enzyme/inhibitor complexes. Similarly, the structure of a fungal al,2-mannosidase that trims Man9GIcNAc2 to Man5GlcNAc2 will be determined by X-ray crystallography. The other enzymes are Golgi a-mannosyltransferases required for fungal glycoprotein and cell wall mannan synthesis. The structure of the al,2-mannosyltransferase (Kre2p) from S. cerevisiae involved in both 0- and N-glycan synthesis will be determined by X-ray crystallography. The importance of disulfide bonds and free sulfhydryl groups, as well as the catalytic mechanism, will be studied by mutagenesis and peptide mapping, and the GDP-mannose binding site will be identified by photoaffinity labeling. The recombinant catalytic domain of Ochip, the al,6-mannosyltransferase that initiates mannan synthesis, will be purified for similar structure-function studies. In addition, the yeast al ,2-mannosidase will be used to study a novel mechanism of membrane protein endoplasmic reticulum localization that depends upon recycling from the Golgi mediated by Rerip. The structural studies will be useful for rational drug design. The Class I a-mannosidases are potential targets of antimetastatic, antiviral agents and of inhibitors that can stabilize misfolded glycoproteins in genetic diseases, while the yeast amannosyltransferases are potential targets for the development of antifungal agents.